Usher Syndrome (USH) and non-syndromic retinitis pigmentosa (NSRP) are degenerative diseases of the retina. USH is clinically and genetically heterogeneous and by far the most common type of inherited deaf-blindness in humans. The hearing impairment in USH patients is mostly stable and congenital and can be partly compensated by hearing aids or cochlear implants. NSRP is more prevalent than USH, occurring in 1 per 4,000 individuals. The degeneration of photoreceptor cells in USH and NSRP is progressive and often leads to complete blindness between the third and fourth decade of life, thereby leaving time for therapeutic intervention. Mutations in the USH2A gene are the most frequent cause of both disorders. The range of mutations is spread throughout all 72 USH2A exons and their flanking intron sequences, and contains nonsense and missense mutations, deletions, duplications, large rearrangements, and splicing variants. The by far most frequently mutated exon is exon 13, which contains two founder mutations (c.2299delG (p.E767SfsX21) in Usher Syndrome type II (USH2) patients and c.2276G>T (p.C759F) in NSRP patients). For exon 50, fifteen pathogenic mutations have been reported, of which at least eight are clearly protein truncating. The first deep-intronic mutation in intron 40 of USH2A (c.7595-2144A>G) was reported by Vaché et al (2012. Hum Mutat 33:104-8). This mutation creates a cryptic high-quality splice donor site in intron 40 resulting in the inclusion of an aberrant exon of 152 bp in the mutant USH2A mRNA, and leading to premature termination of translation (see FIGS. 1A and B of WO 2016/005514).
USH and other retinal dystrophies have for long been considered as incurable disorders. However, several phase I/II clinical trials using gene augmentation therapy have led to promising results in selected groups of LCA/RP/USH patients with mutations in the RPE65 and MYO7A genes. The size of the coding sequence (15,606 bp) and alternative splicing of the USH2A gene and mRNA, respectively, hamper gene augmentation therapy, due to the currently limiting cargo size of many available vectors (e.g. adeno-associated (AAV) and lentiviral vectors).
Despite the broad clinical potential of antisense oligonucleotide (AON)-based therapy, it is not frequently used in the vertebrate eye. AONs are small (16-25 nucleotide) polynucleotide molecules that are able to interfere with splicing as their sequence is complementary to that of target pre-mRNA molecules. Upon binding of an AON, the targeted region of the pre-mRNA is no longer available for splicing factors which results in skipping of the exon that is targeted by the AON. Therapeutically, this methodology can be used in two ways: a) to redirect normal splicing of genes in which mutations activate cryptic splice sites and b) to skip exons that carry (protein-truncating) mutations in such a way, that the reading frame of the mRNA remains intact and a (partially) functional protein is made. For the USH2A gene, 28 of the 72 exons can potentially be skipped without disturbing the overall reading frame of the transcript. Both AON-based methods are being successfully applied in patients with severe genetic disorders. Liguori et al. (2016. Hum Mutat 37:184-193) showed that an AON could prevent the inclusion of a 155 bp pseudo exon 50 (PE 50), caused by the c.9959.4159A>G mutation in intron 50, in the mRNA of the USH2A gene.
It is an objective of the invention to provide a convenient therapeutic strategy for the prevention, treatment or delay of USH and/or NSRP caused by the c.7595-2144A>G mutation present in the intron between exon 40 and 41 of the human USH2A gene. It has previously been suggested (Vaché et al. 2012) and thereafter demonstrated (WO 2016/005514) that AONs are able to block the aberrant splicing of USH2A pre-mRNA that is caused by this mutation leading to the inclusion of pseudo exon 40: PE40. Notably, there is a need for further improved alternatives that perform better and that have additional beneficial properties. An objective of the present invention is to provide such alternatives.